Enrichment of the Basic/Cationic Urinary Proteome Using Ion Exchange Chromatography and Batch Adsorption

Abstract

Anionic or acidic proteins are the main compositions of normal urinary proteome. Efforts to characterize human urinary proteome, thus, have focused mainly on the anionic compartment. The information of cationic or basic proteins present in the normal urine is virtually unknown. In the present study, we applied different methods to enrich cationic urinary proteome. Efficacies of these methods were compared using equal volume (1 L) of urine samples from the same pool obtained from 8 normal healthy individuals. Cation exchange chromatography using RESOURCE-S column provided the least amount of the recovered proteins, whereas batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 4.8) provided the greatest yield of protein recovery. The recovered proteins were then resolved with 2-DE (pI 7-11) and identified by peptide mass fingerprinting using MALDI-TOF MS. There were several isoforms of immunoglobulin heavy and light chains enriched by these methods. In addition, three isoforms of interferon alpha-3 (IFNalpha3) and six isoforms of eosinophil-derived neurotoxin (EDN), were also enriched. The enrichment of IFNalpha3 and EDN was particularly effective by batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 6.0). Initial depletion of anionic components using DEAE batch adsorption reduced the recovery yield of these two proteins and did not improve recovery of any other cationic urinary proteins. We conclude that batch adsorption using SP Sepharose Fast Flow beads equilibrated with acetic acid (pH 6.0) is the method of choice to examine the basic/cationic urinary proteome, as this protocol provided the satisfactory yield of protein recovery and provided the greatest amount as well as maximal number of IFNalpha3 and EDN isoforms. Our data will be useful for further highly focused study targeting on cationic/basic urinary proteins. Moreover, the techniques described herein may be applicable for enrichment of cationic proteomes in other body fluids, cells, and tissues.

Human Urine Proteomics: Analytical Techniques and Clinical Applications in Renal Diseases

“…Therefore, this technique has been introduced as a suitable method for phosphoproteome enrichment [ 31 ]. Thongboonkerd et al showed application of SCX for enrichment of the basic/cationic urinary proteome [ 32 ].…”

Identification of low-abundance proteins via fractionation of the urine proteome with weak anion exchange chromatography

“…The end result is more bound proteins being released from the gel resin. Thongboonkerd et al used SP Sepharose Fast Flow Bead (a cationic ion exchange chromatography) to enrich the basic proteins in urine such as eosinophil-derived neurotoxin and interferon alpha [20]. This study used stepwise elution anion exchange chromatography for the fractionation of urine proteins and the enrichment of low-abundance proteins.…”

“…Certain fractionations have been widely used prior to 2-DE analysis in order to obtain more comprehensive information. For example, immunoaffinity subtraction chromatography [17], ligand beads [18], preparative electrophoresis and 2-DE [19], cation exchange chromatography in combination with a batch-absorption method [20], and finally, a commercially manufactured protein depletion kit to remove the six most abundant human plasma proteins (including albumin, transferrin, haptoglobin, immunoglobulin G, immunoglobulin A, and alpha-1 antitrypsin) [21] have all been utilized for this purpose.…”

Protein and peptide fractionation, enrichment and depletion: Tools for the complex proteome

“…These have been extensively used to fractionate proteins from a host of complex biological samples including nipple aspirate fluid from healthy and breast cancer patients [45], brain lysates [46] and vascular smooth muscle pig cells where a protein concentration dynamic range of 10 7 was achieved [47]. Often utilised in combination with other separation technologies such as 2-DE [48], SDS-PAGE [49] or RPLC [50,51], IEC can assist in improving proteome coverage [52] by facilitating the detection of low abundance proteins or targeting subproteomes through enrichment strategies [53]. There are, however, several IEC-associated drawbacks and these include: (i) the large amount of salts in the elution buffer (up to 2 M NaCl) [54], which can lead to irreversible protein absorption to the resin resulting in loss [55]; (ii) the high salt concentrations present in the eluants, which render samples incompatible with techniques such as IEF and MS; and (iii) the need for clean-up steps to remove salts, which can increase the chances of protein loss.…”